Deletion of MIF gene from live attenuated LdCen−/− parasites enhances protective CD4+ T cell immunity

Vaccination with live attenuated Leishmania parasites such as centrin deleted Leishmania donovani (LdCen−/−) against visceral leishmaniasis has been reported extensively. The protection induced by LdCen−/− parasites was mediated by both CD4+ and CD8+ T cells. While the host immune mediators of protection are known, parasite determinants that affect the CD4+ and CD8+ T cell populations remain unknown. Parasite encoded inflammatory cytokine MIF has been shown to modulate the T cell differentiation characteristics by altering the inflammation induced apoptosis during contraction phase in experimental infections with Leishmania or Plasmodium. Neutralization of parasite encoded MIF either by antibodies or gene deletion conferred protection in Plasmodium and Leishmania studies. We investigated if the immunogenicity and protection induced by LdCen−/− parasites is affected by deleting MIF genes from this vaccine strain. Our results showed that LdCen−/−MIF−/− immunized group presented higher percentage of CD4+ and CD8+ central memory T cells, increased CD8+ T cell proliferation after challenge compared to LdCen−/− immunization. LdCen−/−MIF−/− immunized group presented elevated production of IFN-γ+ and TNF-α+ CD4+ T cells concomitant with a reduced parasite load in spleen and liver compared to LdCen−/−group following challenge with L. infantum. Our results demonstrate the role of parasite induced factors involved in protection and long-term immunity of vaccines against VL.

Visceral leishmaniasis (VL) is considered the second most frequent cause of mortality and the fourth most frequent cause of morbidity within tropical diseases, with 20,000-40,000 deaths per year 1 and significant economic impact due to an estimated 2 million disability-adjusted life years lost 2 . Strategies to eliminate VL in endemic areas rely on rapid detection and treatment of VL to reduce the number of human reservoirs, and vector control using indoor residual spraying 3 . However, elimination programs in endemic areas have not consistently met the intended milestones, and the need for potent diagnostic, treatment methods and prophylactic vaccines against VL to ensure long term control and prevent reemergence of VL is recognized 3 . Vaccination against VL is considered feasible since long-term protection is acquired following clinical cure of VL in majority of the cases as evidenced by previous studies in VL endemic areas [4][5][6][7] . In addition, protection in cutaneus leishmaniasis is observed against infection resulting from the process of leishmanization 5 . A broad range of vaccines including recombinant antigen vaccines, heat-killed parasites, adeno-viral vectored vaccines have been tested against VL [8][9][10][11][12][13] . Yet no approved vaccine for human VL exists.
We have previously reported on the safety and immunogenicity characteristics of centrin gene deleted live attenuated Leishmania parasites (LdCen −/− ) as prophylactic vaccines in pre-clinical animal models [14][15][16][17][18][19][20][21] . Gene deleted Leishmania parasites such as LdCen −/− would present a broad repertoire of antigens to generate protective immunity while undergoing limited replication in the immunized host 22 . Since the LdCen −/− parasite have growth defects in amastigote forms, but not in promastigotes 23,24 , this deletion prevents cell division and longterm persistence in animals (mice and hamsters) or in human macrophages ex vivo 23  www.nature.com/scientificreports/ characteristics of Centrin gene deletion were also observed in other species, like L. major, L. mexicana and L. braziliensis [25][26][27] . In pre-clinical studies, vaccination with LdCen −/− proved to be safe, protective and in mice (BALB/c and SCID), hamsters and dogs, after challenge with virulent parasites, in addition to cross-protection in animals challenged with L. braziliensis, L. infantum and L. mexicana [14][15][16][17][18][19][20][21]28 . Previous studies analyzing the protective immune response following immunization demonstrated the central role of long-lasting Th1-type response 4,9,[29][30][31][32][33][34][35][36][37] . Thus, the induction of a Th1 type response has been considered a pre-requisite in attempts to identify molecules of the parasite and adjuvants that would induce this phenotypic profile in vivo models 9 . Since IFN-γ plays an essential role in the activation of macrophages, allowing the elimination of intracellular pathogens and protecting the host cell against infection 38 , its production is considered one of the main objectives in the immunization process against leishmaniasis. Accordingly, the protective immunity induced by LdCen −/− parasites has been shown to be mediated by Th1 dominant multifunctional CD4 + and CD8 + T cell populations that orchestrate the assembling of granulomas in the spleens followed by the parasiticidal activities mediated by nitric oxide 14 . Recent studies also showed that in a centrin deletion mutant of L. major, skin resident TRM (Tissue Resident Memory) cells also play an important role in protection 39 . Towards understanding the immune mechanisms that direct the development of Th1-type response following LdCen −/− immunization, studies investigated early interactions between LdCen −/− parasites and the innate immune cells. These studies revealed reprogramming of M1/M2 macrophages, reconfiguration of the membrane architecture to enhance cholesterol driven fluidity, variable co-stimulatory and co-inhibitory ligands, and chemokine/cytokine signatures and the corresponding regulatory microRNAs enabling the development of Th1 type immune response following LdCen −/− immunization compared to infection with virulent LdWT parasites 14,[40][41][42] . Yet, while the immunological characteristics of the innate and adaptive responses showed significant differences between LdCen −/− and LdWT infections, the parasitic factors that drive these changes remain to be understood. As the mediators of protection, the characteristics of CD4 + and CD8 + T cell memory subpopulations and their role in prophylaxis or treatment [43][44][45][46][47][48][49][50][51] has been studied thoroughly. A study evaluating different types of T cells in visceral leishmaniasis, including memory T cells without identifying subtypes, demonstrated a lower number of memory T cells in patients with VL compared with treated or asymptomatic patients 52 . Different combinations of effector memory (T EM )/central memory (T CM ) T cells have been shown to be important in inducing protection against secondary infections by Leishmania [53][54][55][56][57] . Thus, successful vaccination must be based on a low dose of antigen, to allow slow replication of effector cells and favor differentiation of memory T cell populations 57,58 .
Studies in Leishmania and Plasmodium identified a parasite-encoded ortholog of a cytokine macrophage migration inhibition factor (MIF) that has been shown to affect the macrophage apoptosis, activation signals, CD4 T cell apoptosis, and CD4 effector T cell responses [59][60][61][62] . Evidence indicates that Leishmania encoded MIF cytokine may drive an inflammatory environment that is detrimental to the host response 63 . Deletion of MIF in Leishmania major parasites showed that antigen presenting, T cell priming and the IFN-γ, IL-7R production by CD4 T cells were significantly affected due to the loss of MIF genes 63 . Studies in Plasmodium berghei showed that Plasmodium encoded MIF enhanced inflammatory cytokine production and induced antigen experienced CD4 + T cells to develop into short-lived effector cells rather than memory precursor cells. The short-lived effector CD4 T cells were more readily eliminated by Bcl-2-associated apoptosis, resulting in decreased CD4 T-cell recall responses against challenge infections 61 . Thus, to investigate the role of Leishmania encoded MIF in generating effector and memory T cell populations following LdCen −/− immunization, we created additional deletion of MIF genes (MIF1 and MIF2, tandemly arranged MIF genes) in the vaccine strain and analyzed the long-term immune response. Leishmania MIF has been shown to interact with its receptor CD74 and exhibit an anti-apoptotic activity that may facilitate the intracellular persistence of the parasite in macrophages 59 . Thus, the MIF deletion would likely increase the long-term immune response that is essential for a successful VL vaccine.
We have evaluated the profile of memory T cells after immunization with the Leishmania donovani doubleknock out parasites (LdCen −/− MIF −/− ) and single attenuated (LdCen −/− ) parasites. Immunized mice with both types of parasites demonstrated a strong immune response, capable of inducing T CM populations following immunization. The changes in the profile of T EM populations after challenge were also observed, resulting in significant cross-protection against infection with virulent L. infantum parasites.

Results
LdMIF proteins induce inflammatory cytokines. Leishmania donovani genome contains two tandemly arranged copies of MIF genes that share significant homology (Fig. 1A). The two ORFs corresponding to MIF genes were PCR amplified and used to produce recombinant proteins. Coomassie stained gel showed LdMIF1 and LdMIF2 proteins were purified to a high degree (Fig. 1B). To test whether LdMIF proteins induce inflammatory cytokines similar to other parasitic protozoa, purified recombinant LdMIF1 and LdMIF2 proteins were incubated with murine BMDCs. Culture supernatants showed significant induction of TNF-α in presence of either LdMIF1 or LdMIF2 proteins relative to untreated control (Fig. 1C). Similarly, BMDC cultures incubated with rLdMIF1 and rLdMIF2 (not shown) also showed an induction of IL-12 in a dose dependent manner (Fig. 1D). Thus, we observed that LdMIF1 and LdMIF2 proteins were able to induce a proinflammatory response.

Deletion of MIF genes does not affect the amastigote proliferation.
To test the effect of the LdMIF induced inflammatory response in immunogenicity, we prepared MIF deletion mutants. The two tandemly arranged copies of LdMIF genes and the genomic context are shown ( Fig. 2A). The gene replacement construct contained either Blasticidin or Puromycin targeted deletion of both copies of MIF genes including the intergenic region. The MIF genes were deleted in a sequential transfection with Blasticidin and puromycin  Fig. 2B).
To test whether deletion of MIF genes affects the parasite fitness and growth as amastigotes, murine macrophages were infected with LdWT, LdMIF −/− parasites (Fig. 2C). Results showed that deletion of MIF genes does not affect the growth of amastigotes as their growth paralleled that of LdWT parasites (Fig. 2D). To test if MIF deletion affects the parasite replication in vivo, mice were infected with LdMIF −/− parasites and compared to mice infected with L. donovani WT. After 4 weeks, splenic burden was measured. Results showed that LdMIF −/− infected mice presented parasites in spleen (Fig. 2E). Deletion of MIF in L. donovani parasites does not affect replication. However, we observed less parasites (not statistically significant) in spleen of LdMIF −/− infected mice. It could indicate that the mutant parasites grow slower than wild type strain but further studies are necessary.
MIF deletion results in reduced CD4 + T cell apoptosis. To test whether MIF deficiency results in reduced inflammation and thus diminished T cell apoptosis in vivo, we infected Balb/C mice with LdWT, and LdMIF −/− parasites. T cell apoptosis in the spleens of the infected mice was monitored by flow cytometry as shown (Fig. 3A). Results showed that the CD4 + T cell apoptosis (Annexin-V + ) was significantly reduced on days 5 and 9 of the LdMIF −/− infection compared to LdWT infection (Fig. 3B,C, respectively) corresponding to the expansion phase of the T cell responses post-infection. No significant difference was observed in the Annexin + CD4 T cell population on day14, that corresponds to the post-contraction phase (data not shown). Similar differences in CD8 + T cell populations were also evident to a lesser degree in LdMIF −/− infection (Fig. 3D,E). Correspondingly, IFN-γ production from the splenocytes of LdMIF −/− infected mice showed a significant reduction compared to LdWT infection after 5 (p = 0.01) and 7 (p = 0.001) days (Fig. 3F). Immunohistochemistry of the spleens from LdWT, and LdMIF −/− 9 days post infection showed the presence of TUNEL + cells in LdWT but much less in LdMIF −/− infection (Fig. 3G) indicating that deletion of MIF promotes the survival of T cells in the infected spleens consistent with our flow cytometric analysis.  (Fig. 4C). Anti-Leishmania vaccines induce humoral immune response that are often used as a surrogate of adaptive immunity which is the main driver of protection. The ability of the attenuated parasites to induce IgG Total , IgG 1 and IgG 2 antibodies against soluble antigen of Leishmania infantum, after immunization or challenge, was investigated ( Fig. 4D-F). The profile of antibodies was measured 4 weeks postimmunization (4wpi), as well as 4, 8 and 12 weeks after the challenge (wpc). Immunization with the doubleattenuated strain LdCen −/− MIF −/− increased the secretion of IgG Total at 8wpc (p < 0.01) (Fig. 4D) and IgG 1 at 4wpi and 8wpc (p < 0.01) (Fig. 4E). In addition, both LdCen −/− and LdCen −/− MIF −/− attenuated parasites were able to induce higher levels of IgG Total (4wpc and 12wpc) (p < 0.01) (Fig. 4D), IgG 1 (4wpi, 4 and 12wpc) (p < 0.01, p < 0.001 and p < 0.01, respectively) (  infantum WT) had an effect on T cell function, we first evaluated the capacity of splenocytes T cells to proliferate upon specific SLA stimulation. The profile of proliferation was evaluated at 4wpi, 4wpc and 12wpc. For this, we used flow cytometry to measure the mean intensity fluorescence of Ki67 in CD4 + and CD8 + T cells (Fig. 5). We observed a significant increase of proliferation of CD4 + and CD8 + T cells on groups immunized with LdCen −/− at 4wpi (p < 0.01) (Fig. 5A) and of CD8 + T cells on group immunized with LdCen −/− MIF −/− at 4wpi (p = 0.024) (Fig. 5B). For CD4 + T cell, in vitro stimulation with SLA did not increase proliferation after challenge. In fact, after 4wpi there was a decrease in proliferation of CD4 + T cells under SLA stimulation (p < 0.01) (Fig. 5A). In CD8 + T cell subpopulation, double-attenuated strain LdCen −/− MIF −/− decreased proliferation at 4wpi (Fig. 5A), but after the challenge with L. infantum this group of cells was able to proliferate in vitro under SLA stimulation (p < 0.01) (Fig. 5B).     (Fig. 8A,B). We observed no statistical differences between groups in 12wpc for CD4 + and CD8 + T CM cells (Fig. 8A,B). CD4 + and CD8 + T EM early showed a decreased frequency of cells in LdCen −/− MIF −/− group compared to PBS group after 4 weeks post-immunization (p < 0.05 and 0.01, respectively) (Fig. 7A,B). After challenge, we observed that the frequency of CD4 + and CD8 + T EM early cells significantly decreased in immunized groups compared to PBS group in 4wpc (LdCen −/− : p < 0.01 and 0.05, respectively) (LdCen −/− MIF −/− : p < 0.05 for both), but not in 12wpc (Fig. 8C,D). We also observed that frequency of CD4 + T EM late cells decreased in LdCen −/− MIF −/− group compared to PBS group after 4 weeks post-immunization, but no statistical difference (Fig. 7A), while frequency of CD8 + T EM late cells was similar between groups in 4wpi (Fig. 7B). After challenge, immunization with LdCen −/− MIF −/− followed by 4wpc showed an increase of percentage of CD4 + T EM late cells compared to PBS and LdCen −/− groups (p < 0.05) (Fig. 8E). No differences were observed in frequency of CD8 + T EM late cells between groups post challenge (Fig. 8E,F).

Intracellular cytokines expression by T cells in LdCen
We also evaluated the proportion of memory CD4 + and CD8 + T cells subpopulations 4 weeks post-immunization ( Fig. 7.1 and 7 Table 1). The data was represented as parts of whole, to show the fraction of total the subpopulations inside CD3 + CD4 + and CD3 + CD8 + T cells. Mean values of each subpopulation and timepoint is plotted, and the scientific graphic program considers the sum of the populations as 100%. Post-immunization, we observed a similarity between PBS and LdCen −/− groups, once they demonstrated high proportion of CD4 + (Fig. 7.1A,B) and CD8 + (Fig. 7.2A  www.nature.com/scientificreports/ and 7.2C). After 4 and 12 weeks of challenge, the profile has changed, we observed a predominance proportion of CD4 + and CD8 + T EM late cells (Fig. 8.1C-F) and CD8 + (Fig. 8.2C-F) in LdCen −/− MIF −/− immunized group. After 12 weeks of challenge, the groups showed a predominance of distinct memory T cells subpopulations. We showed that CD4 + (Fig. 8.1E,F) and CD8 + (Fig. 8.2E,F) T EM late cells proportion was higher in both immunized groups compared to PBS group, where CD4 + and CD8 + T CM cells were majority (Fig. 8.1D and 8.2D).

Cytokines secretion by memory T cells.
Previous studies have suggested that the balance of pro-and anti-inflammatory cytokines may be associated with protection against leishmaniasis [64][65][66] . IFN-γ, IL-12/IL-23p40, TNF-α, IL-17A, IL-10 and IL-4 production was assessed in the subpopulations of stimulated memory CD4 + and CD8 + T cells by flow cytometry, in splenocytes isolated from mice immunized with LdCen −/− and LdCen −/− MIF −/− , at 4wpi and 12 wpc (Fig. 9-9.1-T CM , 9.2-T EM early and 9.3-T EM late ). The analyses strategy can be observed in supplementary Fig. 3. p values can be found in supplementary Table 2 (T CM ), supplementary Table 3 (T EM Early ), and supplementary Table 4 (T EM Late ).
Interestingly, at 12wpc the cytokine profile for T CM cells was different. In CD4 + T CM cells subpopulation, LdCen −/− MIF −/− group showed a higher percentage of IL-12p40 expressed by CD4 + T cells, compared to LdCen −/− group (Fig. 9.1C). Meanwhile, CD8 + T CM cells showed a higher percentage of IFN-γ, IL-12p40, IL-10 and IL-4, compared to PBS group (Fig. 9.1D), indicating that the manipulated Leishmania can address differential activation in different T cell subtypes.
Regarding the expression of cytokines by T EM early (Fig. 9.2), at 4wpi, LdCen −/− MIF −/− group showed an increase in all evaluated cytokines produced by T cells compared to PBS and LdCen −/− groups ( Fig. 9.2A,B). In CD8 + compartment such difference was observed in IFN-g, TNF-a, IL-17A and IL-10 at 4wpi (Fig. 9.2B). Interestingly, at 12wpc significant differences between immunized groups were only seem against PBS group for all cytokines, but IL-10 by CD4 + T cells ( Fig. 9.2C,D).

Discussion
The development of immunity against Leishmania induced by vaccination is widely discussed due to the complexity and antigenic variability of the parasites. Among the models used to study the effect of novel vaccines candidates against Leishmania infection, BALB/c and C57BL/6 strains are widely used on those studies 67,68 . The reasons to choose between both strains are: BALB/c mice show an intermediate pattern of activation that favors parasite persistence and chronicity of the disease, while C57BL/6 mice show a classical pattern of activation associated with resolution of the disease 69 . Vaccination using attenuated forms of parasites allow the immune system to interact with a wide repertoire of antigens 69 , inducing a more robust and complete response when compared to recombinant antigen vaccines 70 because of the presentation of complete array of parasite antigens to the host immune system. In this study, we evaluated a vaccine against visceral leishmaniasis consisting of a double genetically deficient Leishmania donovani for the Centrin 1 gene and macrophage migration inhibitory factor (LdCen −/− MIF −/− ). The centrin1 gene is associated with cell division, specifically affects the proliferative capacity of amastigote forms that replicate within macrophages but does not affect the replication of promastigote forms. The safety of infection with LdCen −/− has been previously demonstrated in mice, hamsters and dogs [14][15][16][17][18][19][20][21] . The MIF gene encodes a lymphokine involved in cell-mediated immunity, inhibiting the proliferation of memory cells 71 . It is demonstrated that parasites express MIF-like genes and could interact functionally with the MIF receptor  www.nature.com/scientificreports/ (CD74) acting as evasion mechanism for infection success 59,[72][73][74][75][76][77] . We hypothesized that the use of a parasite knockout for MIF ortholog could induce a long-term memory response, activation and proliferation of B and T cells, inducing cytokines production, and resulting in a cross-protective immunity against L. infantum. Studies of MIF deletion in Leishmania major showed that mice infected with such parasites presented a reduced ability of the parasite to activate antigen-presenting cells, and consequently a reduction in T-cell priming 60 . Also, mice infected with MIF deleted parasites presented a reduction in generation of inflammation and effector CD4 + T-cell. Effector CD4 + T cells from MIF deleted parasites from infected mice showed a profile of decreased apoptosis, and increased expression of IFN-γ and IL-7R, suggesting that the expression of the orthologue MIF promotes parasite persistence by manipulating the host response to increase the exhaustion and depletion of protective CD4 + T cells 60 . However, while low dose infection with wild-type L. major parasite is known to result in the acquisition of long-term protection, a process known as leishmanization, after resolution of cutaneous lesions, a persistent infection with L. major is established in the host. Thus, this practice has been discontinued due to safety concerns, mainly related to the pathogenicity eventually resulting from uncured lesions, persistence of the parasite in the lesion and reduced vaccine effect (immunosuppression) in patients immunized with vaccine against diphtheria, Bordetella pertussis and tetanus 4,5,11 . Based on this concept, Zhang et al. 25 , using a CRISPR genome edited L. major strain (LmCen −/− ), demonstrated that wildtype L. major infected/healed (leishmanization) and LmCen −/− immunized mice presented high percentage of T CD4 + memory cells producing IFN-γ. The low levels of persistent antigens may be important for maintaining long term protection profile, as the generation of IFN-γ producing CD4 + T effector populations 25,78 , despite the difference between the survival profile of parasites used in leishmanization and immunization with attenuated parasites. Independently T CM and skin resident T RM memory T cells were also shown to play a role in protection in L. major mouse models 39,79 . Thus, it would be hard to discriminate the role of memory cell populations in a L. major infection model due to the presence of memory and effector populations and the kinetics of their actions following a challenge infection. Therefore, LdCen −/− MIF −/− parasites provide an ideal vector to test the role of memory cells in protection considering all characteristics before described here. The role of the anti-leishmanial antibody response seen in VL is unclear. Some authors have suggested that the presence of anti-leishmanial antibodies could be predictive of disease [80][81][82] . In the other hand, it has been demonstrated that antibodies against Leishmania persist for a long time (> 15 years) after cure and immunity to VL 83 . Moreover, it has a high prevalence of seropositive healthy individuals in areas endemic for VL 84 . Here, we demonstrated that immunization with the double-attenuated strain LdCen −/− MIF −/− increased the secretion of IgG, IgG 1 and IgG 2 (regulated by IL-12 induced production of IFN-γ 85,86 ), being able to activate B cells as late as 12 wpc.
The protective immune response against Leishmania is mainly mediated by T cells 87 , and the proliferation of these cells is an important indicator of vaccine immunogenicity tested in mice and dogs [88][89][90] . Our previous work  www.nature.com/scientificreports/ has shown that immunization with LdCen −/− , in dogs and mice, induced T cell proliferation upon stimulation with Leishmania antigens 15,16,28 . Consistent with our previous reports, our new study presented here shows that it also happens in mice for LdCen −/− MIF −/− immunization. Naïve CD4 T cells post-activation undergo programming for inducible production of cytokines leading to generation of memory cells with various functions. The importance of the Th1/Th2 balance in the outcome of leishmaniasis has been demonstrated by many studies [91][92][93][94][95] . Peine and collaborators 96 described that hybrid Th1/2 cells arise naturally during parasite infections and that the two opposing differentiation programs can stably co-exist in resting memory for months, demonstrating a cell-intrinsic self-limiting mechanism that can prevent excessive inflammation. These facts corroborate our findings with an increased mixed cytokines production by T cells after immunization and a persistent of IL-12 production by double-mutant parasites after challenge leaded to a protective profile seem in this study.
One of the most crucial aspects of vaccination is the understanding of the T cell memory profile required to obtain effective vaccines against parasites and virus. T cell memory is the ability of a population to respond to a challenge, recognizing an antigen by its receptors (TcRs). T cell response occurs after the initial exposure to the antigen, by proliferating and/or expressing molecules capable of mediating an effector reaction. A pertinent memory is associated with protection against infection and/or disease when challenged with a pathogen in experimental model 97 . Briefly, naïve T cells (TN) respond to antigenic peptides complexed to major histocompatibility complex (MHC) molecules on antigen-presenting dendritic cells (DCs) after an initial antigenic exposure and priming. After priming, part of those cells proliferates and become effector-memory T cells (T EM ), losing the molecules and CD45RA in the process, and being held back in secondary lymphoid tissues (SLT) 98 . Prompt protection is conferred by tissue resident or circulating effector memory T cells (T EM ) that survey frontline barriers and affected tissues for incoming pathogens and exhibit immediate effector role upon antigen recognition. Another portion of primed cells become central memory T cells (T CM ), responsible for recalling responses and to patrol the T cell areas of secondary lymphoid tissues, where they can quickly proliferate in response to antigens 98 . Those circulating quiscent cells are a provision that can respond to the re-encounter with an antigen within SLT by proliferating and differentiating into T EM and T Eff cells over the course of few days. Central memory T cells (T CM ) are considered better applicable to protect against pathogens with longer incubation periods, such as protozoans 97 . Studies testing single or polyproteins recombinant proteins from Leishmania showed that immunization successfully generate antigen-specific cells that exhibit characteristics of T CM , cytokine production upon antigen re-exposure and increased Th1 response upon challenge compared to nonimmunized animals 43,[99][100][101][102] . In addition, it has been demonstrated that parasites can use MIF ortholog to actively modulate the host immune response, preventing the development of effective memory CD4 + T cells 61 . Our data demonstrated that immunization with double-attenuated parasites induced T CM cells, while PBS control and LdCen −/− groups presented high percentage of T EM cells after immunization. Interestingly, the percentage of T CM cells of LdCen −/− MIF −/− group after challenge decreased while T EM Late cells increased. It suggests a conversion of the memory subtype using the double-deletion mutant parasites. Thus, the deletion of MIF gene in LdCen −/− attenuated parasites could yield a long-lasting immune response, suggested by the increase of T CM cells after immunization.
The evaluation of parasite load allows us to visualize not only number of parasites, but also viability of the parasite. The level of parasite burden in spleen and liver observed in both LdCen −/− and LdCen −/− MIF −/− immunized groups is decreased compared to the positive control (animals immunized with PBS and challenged) group at 12wpc, suggesting a robust degree of protection. Therefore, the protection obtained in the present study confirms the ability of LdCen −/− MIF −/− and LdCen −/− vaccines to limit parasite replication and prevent severe disease after challenge. Okwor and colleagues 103 , evaluating the differences in the immune responses to live and killed L. major in experimental model, have demonstrated that both are qualitatively different. The data demonstrated that live attenuated parasite induced strong and durable protection against virulent secondary challenges 103 , indicating that is a good way to achieve protective immunity against Leishmania infection by vaccination. In our previous work, vaccination with the attenuated parasite proved to be safe, protective and persistent in mice (BALB/c and SCID), hamsters and dogs, after challenge with wild forms, in addition to cross-protection in animals challenged with L. braziliensis, L. infantum [14][15][16][17][18]27,40 . In addition, LdCen −/− MIF −/− immunized mice appear to clear infection in spleen and liver sooner than those immunized with LdCen −/− parasites.
Overall, the results indicate that the combination of deletion of Centrin and MIF genes produced a prominent immunological response, inducing central memory T cells (long-term immune response) after immunization, T and B cell activation, balanced cytokine production and protection against challenge with wild type strain. Despite the deletion only for MIF gene does not seem to affect growth or replication of the parasite, it does seem to affect the virulence factor. These finding points to the fact that the induction of the profile of memory cells are necessary for a protective response and provide novel insights into developing vaccines against pathogens.
The results indicate that LdCen −/− MIF −/− attenuated parasites are potential candidates for the development of an attenuated vaccine against leishmaniasis.

Methods
Expression of MIF proteins in E. coli. Leishmania  www.nature.com/scientificreports/ (0-100 ng/ml) for 24 h and IL-12 production in the culture supernatants was measured by ELISA. The experiment was performed four times (n = 3/replication), and cells were pooled.   www.nature.com/scientificreports/ promastigotes of LdMIF −/− or LdWT parasites in 10 μl PBS. The mice, with 5-7 weeks of age were divided into three groups (8 animals per group/time). LdCen −/− and LdCen −/− MIF −/− groups received intravenously 3 × 10 6 LdCen −/− or LdCen −/− MIF −/− promastigotes at stationary phase, respectively. Control group received PBS alone. Four weeks after immunization, all animals (including PBS group) were challenged with 3 × 10 6 L. infantum parasites. The immunological parameters were measured 4 weeks post-immunization (4wpi), 4, 8 and 12 weeks (wpc) after the challenge with 3 × 10 6 of stationary phase promastigotes of L. infantum intravenously, as demonstrated in Supplementary Fig. 1. Regarding antibody response, T cell proliferation, intracellular cytokine measurement, subtype of memory and parasite load experiments, we performed the experiments three times. The BALB/c mice (n = 8/group/time point, three replicates). were individually assessed, using the same animals for all those experiments mentioned before.
Antibody responses. Antigen-specific IgG Total , IgG 1 and IgG 2 levels were measured by indirect ELISA 64 .
Briefly, 96 wells micro titer plates (Nalgen Intl., USA) were coated overnight with 5 µg/mL of SLA. For IgG Total , IgG 1 and IgG 2 analysis, sera were added at a 1:100 dilution. Peroxidase-conjugated rabbit anti-mouse IgG Total (1:3000), IgG 1 (1:2000) or IgG 2 (1:1000) antibodies were added for 1 h. The reaction was developed using TMB (Sigma, USA) and H 2 SO 4 stop solution was used. Absorbance was measured on VersaMax 340PC microplate reader (Molecular Devices, USA) at 450 nm. In vitro proliferative response of lymphocytes. Splenocytes were isolated as described above. After 72 h incubation, cell proliferation analysis was performed on splenocytes labeled with Ki-67 APC (clone16A8, Biolegend, CA) essentially as described above, and analyzed the stimulation index using monoclonal antibodies CD3, CD4 and CD8. In this sense, proliferation responses were expressed in terms of stimulation ratio that was calculated as: mean proliferation response of cultures stimulated SLA L. infantum/mean proliferation response of unstimulated cultures as described previously 15 .
Determination of parasite burden. At 4 weeks post immunization, and 4-, 8-and 12-weeks post challenge, the parasite load was measured in the spleen and liver by the limiting dilution assay as previously described 105 .
Statistical analysis. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software Inc, USA). Non-parametric Kruskal-Wallis test followed by Dunns test was used to compare data from all three groups (LdCen −/− , LdCen −/− MIF −/− and PBS). Differences were considered significant when a p value ≤ 0.05 was obtained.

Data availability
The datasets used and/or analyzed during the current study and supporting the conclusions of this article are included in this article. These datasets are also available from the corresponding author on reasonable request.